TW200825100A - Protein and nucleic acid for glutathione-dependent formaldehyde dehydrogenase (GFD), alcohol dehydrogenase and S-nitrosoglutathione reductase from Antrodia camphorata, manufacturing method and uses therefor - Google Patents

Abstract

The invention is related to protein and nucleic acid for glutathione-dependent formaldehyde dehydrogenase (GFD), alcohol dehydrogenase (ADH3) and S-nitrosoglutathione reductase (GSNOR) from Antrodia camphorata (A. cam.) comprising protein containing trifunctional activity (GFD, ADH3 and GSNOR) from A. cam. which have amino acid sequence listed in SEQ ID NO:.1 and further comprising nucleic acid encoding said amino acid sequence. The invention is further related to manufactruing method and uses for said protein or said gene of detoxification enzyme from A. cam.

Description

200825100 VII designated representative map: _: a) The representative representative picture of this case is: sequence identification number L Han i" map, Mi 1D N0: 1, z) 〇 \ (ii simple symbolic description of the symbol of this figure: The present invention relates to a protein of Ganoderma lucidum GFD, an amino acid, and a nucleic acid thereof, a nucleic acid, and a performance vector, host cell, and the like, which can be used for the production of the protein. Manufacturing method, and pharmaceutical composition containing the protein. [Prior Art] Ganoderma lucidum (3) see also known as burdock, burdock mushroom, : The guide indicates that it is very effective for the treatment of liver tumors and cervical tumors (1,3-4) ), because it is only in the mouth of the burdock; ^ growth, so the folks regard the 樟 为 as a treasure. Because 樟 是 is the only 匕 匕 匕 樟 樟 樟 樟 樟 樟 樟 大 大 大 大 大 大 大 大 大 大 大 大 大 大 樟 能 能 能 能 , , The physiological function of the genus, the most common application of traditional Chinese medicine to the traditional abdominal therapy is the application of food, abdominal abdomen, 4 lower abdomen [5 pain, skin pain and liver cancer and other symptoms. In the physiologically active ingredients of the curtain, the antioxidant is particularly affected. Pay attention to (1), so aim at it The analysis of antioxidant components, the antioxidant component, was studied in detail in deep culture, and it was pointed out that in the extract of Ganoderma lucidum (Four), the three-poster has an inhibitory function on the over-gasification of lipids. It plays an extremely important role in scavenging oxygen free radicals. Because it contains a large number of complex antioxidant components, it can protect normal cells or cancer cells by improving the effect. The machine has also been researched and put into practice. The mycelium of A. sinensis mycelium has a strong cytotoxic effect on the liver tumor cell line HeP G2 at the low dose of 7 200825100, followed by water extraction, but The fermentation filtrate was almost non-toxic. The mice were tested in C57BL/6 and BALB/c. The results showed that TNF-α, IFN-T and IL-2 were significantly increased in the blood of rats, while 11^4 and IL were significantly increased. -10 is not significantly different from the control group. In addition, the mycelium fermentation filtrate of Antrodia camphorata has the effect of protecting its liver function by its ability to resist oxidation and scavenging free radicals. Handling can avoid human beings The consumption of GSH and ATP in red blood cells is cytotoxic to leukemia leukimia HL-60 cells, indicating that the mycelium of Antrodia camphorata may have anti-oxidant and tumor-inhibiting effects. Antrodia is very effective in the treatment of liver function diseases. The polysaccharide has a significant inhibitory effect on the hepatitis B virus. Among the published mushroom polysaccharides, Antrodia camphorata is the first species to be confirmed to have a direct inhibitory effect on the hepatitis B virus. There are few reports on the research of the carcass entity, and its curative effect should be higher than that of the mycelium, so the fresh amaranth fruit body material is used as the material.

Glutathione-dependent formaldehyde dehydrogenase (GFD) belongs to alcohol dehydrogenase 3 (ADH3), which mainly decomposes formaldehyde, alcohol and S-nitrososylated proteins, and plays an important role in detoxification and maintenance of health. (7-15) 〇GFD can prevent formaldehyde and protein and nucleic acid from reacting and harming organisms. In plants, it can also metabolize formaldehyde by GFD. (8) 'N0 has many physiological functions, one of which is information transmission, which can transmit Information is based on protein nitration (S-nitrosoylation) or not to achieve the regulation of metabolic enzymes, structural proteins or translation factors. In recent years, it has been found that GFD also has a reduced nitrite 8 200825100 (S-nitrososylation), especially nitr〇s〇giuta let 〇ne (GSN〇), thus assisting 濩 濩 organisms from nitrosaHVe stress (n), There have been no reports of fungi (fungus), especially Taiwan's Antrodia. GDF can also metabolize higher concentrations of alcohol (7). Therefore, Ganoderma lucidum GFD from Taiwan has three kinds of detoxification functions (to remove a jun, alcohol and reduce nitrated substances), which is the first discovery. GFD-related genes often differ in their stability and activity due to different sources, but little is known about the GFD gene products of G. chinensis. Even other organisms have been reported in recent years, so it is worthwhile to Ganoderma lucidum fruiting body GFD. Genes are systematically studied. The RNA was extracted and the cDNA was synthesized by using the fresh amaranth fruit body from Lugu. The EST sequence information was used to design the primers, and the GHD DNA was amplified by PCR using the cDNA of the Aster sinensis as a template. The DNA of the GHD fragment of the Ganoderma lucidum fruiting body was screened, and the cDNA of the full-length GFD was finally obtained by 5fRACE and 3fRACE. This is followed by attachment to a phenotypic vector, implantation of yeast for mass production and purification. The GFD gene product is a protein with a molecular weight of 40 kDa. How to implant eukaryotic cells is another test. Therefore, if several amino acids (Tat) are attached to the N-terminus, the Tat-GFD product will be easily implanted into eukaryotic cells. To play its function. It is worth applying to healthy foods or added to the scalding cream to break down the damage caused by N0. SUMMARY OF THE INVENTION The present invention provides a protein and nucleic acid of Ganoderma lucidum GFD, a vector for producing the protein, a host cell and method, and a composition containing the protein. The present invention is selected from the fresh A. camphora fruiting body and selected to express GFD. The protein removes formaldehyde, alcohol and reduces the nitrated material and is therefore worthy of being applied to health foods or added to scalding creams. 9 200825100 The white matter of the present invention relates to the Gfd protein, and the selection thereof comprises the sequence identification number: the amino acid sequence of the sequence identification number i [SEQ ID N〇: 1] shown in 媢 is according to the present invention. Similarities to the functions of the decamera, phage and reduction-confirmed functions of the Ganoderma lucidum GFD protein f and its gene of the present invention are also included in the scope of the present invention. As used herein, the term "speech", "alcohol, and sulphate" is a protein having the same function as the nitrating, alcoholic, and reduced nitration of the Ganoderma lucidum GFD protein of the present invention. Preferably, the protein of the invention comprises an amino acid sequence as set forth in Sequence Identification 1, 2. SEQ ID NO: 1, 2. According to a preferred embodiment of the present invention, a protein shell having a deformed, alcoholic, and reduced terracotta having a sequence-recognition amino acid sequence is selected which substantially comprises a curric acid. The (g) sequence of the G. sinensis gfd amino acid sequence of the present invention and other species is ton, and the present invention (A) belongs to (4). In addition, the present invention also found that Ganoderma lucidum GFD protein f has good specific activity, thermal stability, protease stability and chemical stability. The protein has the following characteristics: 2 = after C heating - can still maintain a relatively high (_ activity, after 7HU of interest treatment, its activity is not shadow u 8 degree of imidazole treatment, activity can still be dimensional · Intestinal trypsin (caffeine _η) or tryptone ^ ^ which amount 1/2 〇 try try try try try try try try try try try try try try 371 371 371 371 371 371 371 371 371 371 371 371 371 371 371 371 371 371 371 371 371 371 371 Many knots found no significant changes in protein or activity. In addition, according to the present invention, the preparation of Ganoderma lucidum GFD is carried out under the condition that the nucleic acid encoding the protein of the present invention is cultured to recover the protein. Xinbei Tu, Tian Feihan 200825100 Nucleic Acids The invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a G. sinensis GFD amino acid sequence having SEQ ID NO: 1 (see, Figure 1). The phrase "nucleic acid molecule" as used herein is meant to include DNA. Molecules (such as cDNA or genomic DNA), RNA molecules (such as mRNA), DNA or RNA congeners produced using nucleic acid analogs, and derivatives, fragments and homologs thereof. The fumaric acid molecule can be single or double stranded, but is preferably double stranded DNA. The term "π-isolated nucleic acid" as used herein means a molecule isolated from other nucleic acids of natural origin. According to the present invention, a nucleic acid may comprise only a partial fragment encoding the biological activity of Ganoderma lucidum GFD. "Word π fragment" as used herein. This means the portion of the nucleic acid sequence encoding the still biologically active Antrodia camphorata GFD fragment. According to a particular embodiment of the invention, the isolated nucleic acid molecule of the invention has the nucleic acid sequence of SEQ, ID NO: 1, or its degenerate sequence (Wobble hypothesis). In another embodiment, the isolated nucleic acid molecule of the present invention comprises the nucleic acid sequence set forth in SEQ ID NO: 1, which has a full length of 1373 and a translation region of 1134 bp, and which can translate 378 amino acids. The nucleic acid molecules of the present invention have high similarity to sequences from other sources. The Ganoderma lucidum GFD encoded by the nucleic acid of the present invention can decompose formaldehyde, alcohol and reduce the nitrated substance, and provides damage and damage caused by the organism against formaldehyde (formaldehyde, alcohol and N0). Numerous nucleic acids encoding the Ganoderma lucidum GFD protein of the present invention can be prepared based on the degeneracy of the genetic code, as recognized by those skilled in the art. Therefore, for a specific amino acid sequence that has been identified, those skilled in the art can make various nucleic acids by simply modifying one or more codes without changing the amino acid sequence of the Ganoderma lucidum GFD protein. . According to the present invention, the nucleic acid encoding the Ganoderma lucidum GFD protein can be cleavable by standard hybridization and colonization techniques, and the nucleic acid of the present invention can be used from the cDNA library of the fresh A. camphorata fruit body using standard selection and screening techniques 11 200825100 separate from. Amplification of the nucleic acid of the present invention can be carried out according to a PCR amplification technique using cDNA, mRNA or genomic DNA as a template and an appropriate oligonucleic acid primer. The amplified nucleic acid is selected for selection into a suitable vector and identified by DNA sequence analysis. Performance Cutoffs and Host Line Branches The present invention also provides a performance vector comprising the nucleic acid of the present invention. The expression vector of the present invention comprises a nucleic acid sequence encoding the protein as set forth in SEQ ID NO: 1. As used herein, the term "expression carrier" is used to directly express a gene nucleic acid molecule linked thereto. The vector is a nucleic acid that can be self-replicating and expresses a nucleic acid attached thereto. In general, a expression vector for recombinant DNA technology can be used. Usually in the form of a "vector", typically a circular double stranded DNA that is not fused to a chromosome when it is in the form of a vector. A nucleic acid sequence encoding a GFD protein or a functional analog thereof of the present invention can be inserted into an appropriate expression vector to express a biologically active GFD protein. The expression vector is required to contain a 7G piece of transcriptional transcription and translocation necessary for insertion of the coding sequence. A skilled artisan can construct a representation vector containing a protein encoding a GFD protein and appropriate transcriptional and translational control elements using well-known methods. Such methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination techniques. Another object of the invention is to provide a host cell comprising an expression vector comprising a nucleic acid sequence encoding a GFD protein. The term "host cell" as used herein is a host cell that can be infected by a vector, such as a plastid. Many host systems can be used according to the invention to contain and represent sequences encoding a GFD protein. Such host systems include, but are not limited to, A microorganism (such as a bacterium transformed with a recombinant plastid or a expression vector), a yeast (such as a yeast transformed with a yeast expression vector), or an animal cell system. The host cell of the present invention is Escherichia coli and yeast. The invention expresses the cDNA of the GFD protein in the Daphnia bacillus or the yeast system, and can indeed exhibit the active recombinant protein 12 200825100, and obtains the pure GFD protein after rapid purification by the affinity column.

According to one embodiment of the present invention, the Ganoderma lucidum GFD line of the present invention is constructed by using pYEX-Sl• as a phenotype vector; the N-terminal restriction enzyme valence oRI fragment is designed (5, GGAATTCG ATG TCC ACA GTA GGA AAA CC 3,) DNA with PCR derived from the C-terminal primer with 6 His-tag and 7 (9 RI (5, GGAATTCCTA GTG GTG GTG GTG GTG GTG ATG CAT GTC GAC GAC ACA GC 3'), using the cDNA of GFD protein as a template and primer The DNA was sent to E. coli for screening, and the extracted plastids]) να was treated with restriction enzyme and RI, and the resulting fragment was ligated with the same restriction enzyme-treated ρΥΕχ-si _, and sent to yeast yeast, and Medium YPD medium (1% yeast extract, 2% peptone, 2% glucose) was incubated at 30 °C for 5 D (170 rpm), the cells were disrupted, centrifuged at 10, OOOxg for 5 min, and the supernatant was collected and affinity. After the column is rapidly purified. After electrophoresis, the protein was stained. After 10% SDS-PAGE, a clear band was observed at M40kDa, which is the GFD protein. In order to facilitate the passage of the GFD protein through the cell membrane, a Tat containing nine amino acids can be attached to the front end, and the GAD protein DNA attached to Tat can be expressed in the yeast. ^ The present invention also encompasses a pharmaceutical composition comprising the GFD of the present invention. In addition to the protein of the invention, the pharmaceutical composition may contain a suitable pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention can be made in a manner known in the art (e.g., by the conventional method), which includes mixing, dissolving, granulating, tableting, grinding, emulsifying, encapsulating, entrapment, and/or freezing. Dry and other steps. The pharmaceutical composition of the present invention can be administered by any route including, but not limited to, oral, intravenous, intramuscular, intraarterial, transdermal, subcutaneous, intraperitoneal, intranasal or intestinal administration.

实甩A 13 200825100

ΐ / ΛΓ r rf source of Ganoderma lucidum GFD protein f, its activity h its source of GFD ? =: Γ 樟 Γ Γ GF GF GF GF GF GF G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G Value and contribution. The Ganoderma lucidum GFD protein of the present invention can decompose the road, the alcohol and the sputum, and provide the damaging blood caused by the organism (resistance, alcohol and (10)). The GFD protein of the invention is applied to health foods, and the addition of the scald ointment can accelerate wound healing; in the application of plastic surgery and artificial skin transplantation in plastic surgery, gfd can be used

The protein prevents tissue necrosis of the transplanted skin, thereby accelerating the suturing of the transplanted skin. The following examples are intended to further illustrate the invention, but are not intended to limit the scope of the invention, and any alternatives and modifications that are known to those skilled in the art are still within the scope of the invention without departing from the invention. The spirit and purpose. The total RNA of the scorpion scorpion was extracted from 4 g of the scorpion scorpion body in a liquid nitrogen rapid cooling bed. After grinding into a powder in the research spider, the lysis buffer (Novalgen's Straight's mRNA Isolation System) was taken immediately. Mix in a 30 mL centrifuge tube, place at 4 ° C for 5 min, centrifuge at 9, OOO xg for 15 min at 4 ° C, centrifuge and take the upper aqueous layer, approximately 20 mL. The water solution was taken up in a 50 mL falcon and added with 3 mg of magnetight particles. The mixture was placed at 4 ° C. The magnetic holder was loaded with magnetight particles, washed three times with 1.5 mL of wash buffer, and washed at 0. 5 mL with water at 60 ° C. RNA (lOug), add 0·1 vol. 3 M sodium acetate and 0.8 vol. isopropanol at -75 ° C for 20 min, then centrifuge at 1 000 ° C for 15 min at 4 ° C, 70% ethanol Wash a total of three times, dry 14 200825100 After drying, store at -70 ° C for use. Synthesis of Anthraquinone 5'-RACE-Ready cDNA and 3'-RACE-Ready cDNA: - Based on BD Biosciences Clontech^ SMART RACE cDNA • Amplification Kit. 5'-RACE-Ready cDNA : Add all the following reaction solutions to 1.5 mL of microtubes and completed on ice · mRNA (ug/uL) (1 ug) 5,-CDS primer Smart II A Oligo H2〇

1 uL 1 uL

1 uL

2 uL

5 uL 31 2 3 4 5 6 7 8 9-RACE-Ready cDNA: mRN A (ug/uL) (1 ug) 3 9 - CD S primer A h20 Total 15 1

uL 2

1 uL 3

uL 4

5 uL 5 The reaction solution was uniformly mixed and centrifuged slightly and placed in a 72 ° C constant temperature water bath for 6 6 min. After the action, the tube was placed on ice for 2 min. Add 2uL 5X first-strand 7 .... 8 buf fer, 1 uL 20 mM DTT, 1 uL 10 mM dNTP mix, 1 uL powerscript 9 reverse transcriptase in a 42 ° C constant temperature water bath 1. 5 h. After adding 200 uL tricine-EDTA buffer in 72 ° C f-warm water bath for 7 min, store at 200825100 -70 ° C for use. The growth of the GHD gene of Ganoderma lucidum was designed by using the EST sequence information of Antrodia camphorata, using the 5,-RACE-Ready cDNA and 3'-RACE-Ready cDNA as templates to PCR-proliferate GFD-related gene DNA and screen it. The DNA of the GHD gene fragment was further subjected to 5fRACE and 3,-RACE, and finally the full-length GFD cDNA was obtained. GFD cDNA performance

It is developed by PCR, preparation of colloids, electrophoresis, secondary selection, construction of yeast phenotype vector (pYEX-Sl), induction of proteins, and electrophoresis analysis of samples. PCR program The following reaction samples were added to 0.5 mL microtubes and the following reagents were added: template DNA 0.2 ug 5' - sense primer 10 pmol 3'-antisense primer 10 pmol

10XTaq DNA Polymerase Buffer 5uL

6uL 15 mM MgCh

Taq DNA polymerase 2.5 units

10 mM dNTP 1.5 uL

Add sterile water to a total of 50 uL, add 50~100 uL of mineral oil, and perform 25 cycles of PCR (94 °C for 30 200825100 min, 50 °C f〇r 3 min, 72 °C for 1.5 min). After the PCR reaction is completed, DNA can be obtained. The product, after recovery, can be used for further experiments, such as performing a DNA ▲ group and analyzing its sequence. The colloid used in the preparation and electrophoresis of Tengqi was 1.0% agarose gel, which was poured into IX TAE buffer to completely cover the gel. Take 15 uL of PCR product, add 1/10 volume of trace dye, inject into the well, and inject 6 uL of 1 kb DNA marker to understand the size of the DNA sample, and then electrophoresis at 100 volts. When the DNA dye is moved to 2/3 of the gel, the electrophoresis can be stopped. The gelatinized gel was stained with bromine (EtBr) for 15 min, then de-stained in water, and then placed under a UV light source box for observation. Secondary selection 1. PCR product 舆 馑 馑 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主 宿主Make a connection. Take the above-mentioned ligated recombinant DNA, add 18 uL of T0P0 10 active cells, place on ice for 30 min, then place in a 42 ° C water bath for 30 sec for heat shock and immediately place on ice for 2 min, add 150 uL SOC medium was incubated at 37 ° C for 60 min, and evenly spread on a Petri dish containing LB defatted sugar containing 50 ug/mL antibiotic ampicillin. After incubation for 12-16 h at 37 T), select 50- Eighty individual strains were placed on LB agarose of the new ampicillin for further experimentation. 2. Preparation of the radioactive probe of the yeast cell of the cell 17 200825100 After the transfer paper is applied to the surface of the culture dish and positioned with a needle hole, the transfer paper is taken out, and the isolated colony is transferred to the transfer paper. The transfer work is completed. Prepare a shallow pan, overlay 3 mm filter paper, pour the denaturation solution (0.5 M NaoH, 1.5 Μ NaCl), pour the amount to fully wet the 3 mm filter paper, place the transfer paper with the adsorption surface facing up, and put it in the wet Above the filter paper for 7 min, then another shallow pan was taken to neutralize the solution (1 MTris-HCl, 1 M NaCl, pH 7.5) and wet the filter paper for 7 min. After repeating the neutralization step, the transfer paper was placed in an oven and dried at 60-7 (TC. After that, the transfer paper was placed in an ultraviolet cross-linking machine for mesh connection for 15 sec, further radioactive Probes for screening. Preparation of radioactive probes

Voucher nucleic acid (3 pmole/uL) 0· 5 uL 10X τ4 nuclease buffer 3. 0 uL nuclease (12 unit) 1_ 5 uL [r-32p] ATP 3. 5 uL H2O 21. 5 uL Total 30 0 uL Mix well and place at 37 ° C for 30 min. That is, the preparation of the radioactive probe is completed, and the radioactive probe is added to the hybridization solution to screen the desired target gene. 3. Selection of target gene DNA The above transfer paper was taken out, and a lotion prepared with 1.5X SSC and 0.1% SDS was added, shaken at 44 ° C for 30 min, the lotion was poured off, and no radioactivity was added. Probe hybrid 18 200825100 Cross solution (5X SSC, 5X Denhardt, 0.5% SDS, 〇〇叩 / this sperm DNA), this process is called pre-hybridization. Pour out the hybridization solution, add the parent solution containing the radioactive probe, shake it in a 44 C water bath, pour out the hybridization solution containing the radioactive probe, shake it at 44 °c for 15 min with the above lotion, repeat This step is 2-4 times. The transfer paper is dried and subjected to automatic radiographic development. After the film is punched, the positive reaction strain is picked.舆 Purification 舆View The positive reaction strain was picked up in iLB containing ampicillin (5〇 ug/mL) and cultured at 37〇CT for 6-8h. After centrifugation for 3 min at 100 μg, the bacterial cell pellet was obtained, and 2 ul of susceptor (50 mM Tris, pH 7.5, 1 mM EDTA) was added to shake, and the cells were suspended. Add 2 ul of lysate (〇· 2 M NaOH, 1% SDS), mix gently, so that the bacteria can be completely dissolved, and the plastids can be dissolved. Add 200 uL of neutralizing solution (1. 32

Μ vinegar 酉夂 ))) and then centrifuge at 12, OOOxg for 1 〇 min, take the supernatant, add 〇 · 5 mL away from the official (containing DNA adsorption membrane), centrifuge at 12, OOOxg for 1 min, so that DNA can be adsorbed After the membrane was added, 700 mL of the washing solution was added and centrifuged at 12,0 Xg for 2 min. Then centrifuge at 2, 〇〇〇xg for 2 min to remove residual alcohol. Finally, add 5 〇 of this hot water (6 〇t) for 30 sec, centrifuge at 12, 〇〇〇Xg for 2 min, collect and centrifuge. The plastid DNA is used as a plastid restriction reaction and electrophoresis analysis. Take (10) human and appropriate amount of restriction transfer 'plus X reactin buffer, finally add sterile water to make the volume 10 UL 'react at the appropriate temperature for 2 h, analyze with 1% agarose gel to determine the DNA the size of. 19 200825100 5. Sequence Li jl Automated sequencing using the ABI PRISM 377-96 DNA Sequencer. Construction of _1_ phenotype: The expression vector is pYEX-Sl, and the GFD line is constructed with pYEX-Sl as the phenotype vector; the N-terminal restriction enzyme and the ^ Ri fragment are designed. (5, GGAATTCG ATG TCC ACA GTA GGA AAA CC) and C-terminal primers contain 6 His-tag and valence o RI (5, GGAATTCCTA GTG GTG GTG GTG GTG GTG ATG CAT GTC GAC GAC ACA GC 3), which is obtained by PCR using the cDNA of GFD protein as a template and primer. DNA. The DNA was sent to E. coli for screening, and the extracted plastid DNA was treated with restriction enzyme 〇RI, and the resulting fragment was ligated with the same restriction enzyme-treated pYEX-S1, sent to yeast, and YPD medium (1%). Yeast extract, 2% peptone, 2% glucose) 5 D (170 rpm) was incubated at 30C, the cells were disrupted, centrifuged at 10, 〇〇〇xg for 5 min, and the supernatant was collected and quickly passed through the affinity column. After purification. After electrophoresis, protein staining was performed. After 10% SDS-PAGE, a clear band was observed at 40 kDa, which is the GFD protein. More preferably, in order to facilitate the passage of the GFD protein through the cell membrane, a Tat containing nine amino acids may be attached to the front end, and the GAD protein DNA attached to Tat may be sent to the yeast for expression. It was transferred into yeast [trp-ada...ura...] as a performance host. pYEX-Sl (both first and ό RI treatment), the above-mentioned ligated recombinant DM was added to the yeast active cells, and the positive reaction strain was selected for protein expression. In yeast 麄 形 20 200825100 1. To prepare Korean cells (1) Method 1: Select a single colony and completely streak the YPD dish, culture at 28-32 °C for 8~96 h, from bacterial culture medium The surface layer was scraped and the Yeast tern, commercial SC0S mixture was added to 110 pL to bring the bacterial concentration in the test tube to 5 xio7/test tube. (2) Method 2: Select a single fresh colony and add 1~10 mL of YPD medium to incubate at 28~32 °C for 20~30 h. Add the culture solution to 10~100 mL YPD Pei® solution for 8~24h, centrifuge at 2000 X g for 10 min, remove the supernatant, wash twice with sterile water to remove residual YPD medium, and mix the mixture. Into the 110-115 pL SC0S mixture until the bacterial concentration reached 5 x 10 7 / test tube. 2. Prepare the SC0S mixture and add 100 pL SC0S buffer night to 1.5 mL tube, add 5 pL 1 M DTT (stored at -20 °C), add 5 pL ss ΜΑ (stored at -20 °C), add Plasmin DNA (no more than 5 μΙ〇.

• 3. Preparation of dry selective culture I completely dry culture dish can provide good transformation reaction in Yeastern's SC0S transformation system. Therefore, it is recommended to leave the cultured dish to the aseptic table for about 1 hour before the culture is stacked. Wait until it is completely dry. 4. SC0S Korean shape (1) Mix SC0S mixture with yeast suspension cells to make SC0S transmixed mixture 0 21 200825100 (2) Cover the tube and place it at 45·5 °C for 10~60 min. (3) Applying the mixture of the mixture directly to a dry culture dish [YNBD (1.7 g yeast nitrogen base, 5 g ammonium sulfate, 20 g glucose, 15 g agarose in 1 L water) containing 20 ug Trp/mL], The program was completed within 20 sec and the dish was placed at 28-32 °C for 2-4 days. The protein was expressed as a single colony (1~2 mm) in 10 mL YWAAND (1·7 g yeast nitrogen base, 5 g asparagine, 20 g glucose in one L) containing 20 ug Trp/mL medium, using 125 mL flask 'And incubate at 250 rpm in a 30 ° C incubator overnight, and add 5 mL of the culture solution the next day to make ODe. . After reaching 0·1 and continuing to incubate at 30 °C, 250 rpm for 72 h, add 10 mL of YPD and continue to incubate at 30 °C, 250 rpm for 48 h. All the bacterial liquids were centrifuged at 5000 x g for 10 min at 4 ° C. The bacteria were added with 0.5 g glass beads and 2 mL PBS (pH 8.0), the cells were disrupted, centrifuged at 10,000 x g for 5 min, and the supernatant was collected. Add 2 mL of buffer, repeat this extraction step, and finally extract again with 2 mL of buffer. Collect a total of 6 mL of supernatant, which is a crude protein sample. For further electrophoresis, the size of the gel is 10 Cmx8cmx〇.75mm. 1. Preparation of Thunder Diet After the aluminum plate and the glass plate are wiped clean, and the spacer is combined, put into the rubber molding model, and then prepare the colloidal solution according to the dosages listed in the table below. And injected into the model, and then add the appropriate water leveling, about 1 hour, after the formation of the colloid, then inject into the heap 22 200825100 gel, and insert _, to be ready. Take the sample σσ and mix it with the sample loading buffer and inject it into the colloidal hole. The composition is electrophoresed at 100 volts. The native-pAGE takes about 65 minutes, and the SDS-PAGE takes about 130. Colloidal 'C〇〇mass^e biue staining: the gel is next to the dye, shake it evenly for 3〇min, remove the dye, wash it once with water, add _ to the dyeing agent, and defecate for 12 hours. 3. Purification of GFD with 6 His's affinity column purification with Ni2+-nitrilotriacetic acid Sepharose superflow as follows: 3 mL of resin column 'equilibrated with 5 vol·PBS, then 6 mL of crude protein sample Inject and collect about 6 mL of the liquid flowing down. The column was washed with 3 vol. PBS containing 20 mM imidazole, followed by extraction with 1 mM mL PBS containing 100 mM imidazole. A total of 6 I fractions were collected, 2 of which were collected in 3 mL of protein, because Contains imidazole, so it needs to be removed by dialysis. Dialysate: Approximately 3 mL of the sample was placed in the dialysis membrane, clamped with a dialysis clamp, placed in a beaker containing 200 mL of 1/3 PBS containing 1 mM DTT and 5% glycerol dialysate, and dialyzed at 4 ° C for more than 4 h. Repeat this dialysis step to dispense the η step analysis. 5. The protein content of GFD is first prepared by BSA standard curve: egg 23, 200825100 white matter standard (BSA, 0.5 mg/mL), 1, 2, 3, 4, 5, 6 uL The corresponding volume of the sterilized water is 800 uL, and then reacted with a coloring agent of 200 uL for 5 min at room temperature, and the OD595^ is measured and the protein standard curve is determined according to the absorbance. The protein content can be determined from the standard curve by measuring the absorbance value of the harvested sample by the same method. 6. Activity measurement GFD live dyeing: (1) Firstly, the electrophoresis colloid was immersed in an aqueous solution of 70 mM potassium phosphate buffer (pH 8.0), 0.5 M potassium chloride, 4.8 mM formaldehyde and 1 mM reduced GSH at 25 ° C. After shaking for 5 min, different amounts of NAD, nitroblue tetrazolium and phenazine methosulfate were added to make the final concentration of 1.2 mM, 0.4 mg/mL, 0·03 mg/mL, and the colloid was shaken at 37 ° C for 1 h to deionize. After washing the colloid with water, the area showing the purple band is where GFD is active. GSN0R live dyeing: (2) The electrophoresis colloid was immersed in 0.1 Μ potassium phosphate buffer (pH 8.0), 2 mM NADH aqueous solution, placed on ice for 15 min, and the excess buffer was removed. The colloidal coating contained 3 After 15 min of filter paper of mM GSNO, the filter paper was removed and the colloid was observed on the ultraviolet light to see the disappearance of NADH fluorescence. Determination of GFD activity: (3) Partial modification according to Uotila and Koivusalo (13) in 100 uL of reaction solution: 0·25~1 gg GFD, 100 mM potassium phosphate buffer (pH 8. 0), 1 mM HMGSH [GSH And formaldehyde 24 200825100 (1:1) at 25. 5 mL, the amount of GFD was 1. 5 mL, and the amount of GFD was 1. 5 mL, and the amount of GFD was 1. 5 mL. In the test tube, each tube was heated at 50 °C for 0, 2, 4, 8, and 16 min, and then placed on ice for 10% native-gel electrophoresis for protein staining (2 ug) and activity test ( 2 ug). The results show that this is very stable, the time required to reduce the activity by half at 50 ° C is 5 min. The effect of pH is taken separately, in a 1.5 mL tube, a total of 6 tubes, adding different pH Value buffer: 0·2 sodium citrate buffer (pH 2.3, or 5·4), 0·2 M Tris-Hcl buffer (pH 7.8, or 9.0), 0.2 Μglycine NaOH buffer ( pH 10. 4 or 11.2), after reacting at 37 °C for 1 h, placed on ice, and subjected to 10°/〇native-gel electrophoresis for protein staining (2 ug) and activity test (2 ug). This is very stable, showing that the treatment with acid and alkali is very stable, especially to pH 7. 8~pHll treatment of lh has no effect on the activity. SDS effect is taken separately, in a 1.5 mL test tube, a total of 5 Add different amounts of 20% SDS, respectively, so that the final concentration of SDS is 0, 1, 2, 3, 4%. After reacting for 1 h at 37 t, perform 10% native-gel electrophoresis for protein staining (2 ug). And activity test (2 ug). The results show that the treatment of SDS is not stable. 25 200825100 Effect of imidazole

Take appropriate amount of GFD, in a 5 mL tube, a total of 5 tubes, respectively, add different amounts of _ imidazole, so that the final concentration of imi, 〇, 〇. 2, 〇 · 4, 〇. 8, 丨 6 μ, in 37 °C " After the reaction, '10% native-gel electrophoresis for protein staining (2 ug) and activity test (2 ug). The results showed that the treatment with miso was also very stable and must be as high as 0. 8 Μ to inhibit activity (3〇%). Effect of Protease® Take appropriate amount of GFD, respectively, in a 1.5 mL tube, a total of 4 tubes, respectively, add 1/20 of the amount of GFD trypsin or chymotrypsin in 1 ^ -11 (: 1 buffer (011 8 · 5 containing 20 mM CaCl2) reacted at 37 °C for 10, 20, 40 min, respectively, subjected to 10% native-gel electrophoresis for protein staining (2 ug) and activity test (2 ug). The results showed trypsin After 40 min of treatment, the activity is unchanged. Kinetics of kinetic GFD: The Lineweaver-Burke plot of the velocity (l/V〇) for 1/HMGSH is Km = 0_615 mM and Vma^ 128 μΜ/min,the Velocity (l/V〇) is 1.105 mM for 1/NAD and Vmax = 131 μΜ/min.

References (1) Hsiao, G·; Shen, Μ·Y·; Lin, K. H·; Lan, Μ·H·; Wu, L·Y·; Chou, D. S·; Lin, C. H· Su,C·H.; Sheu? JR Antioxidative and hepatoprotective efiFects of Antrodia camphorata extract. •/•Jgrza CTiem. 2 ❶ 03,57, 3302-3308. (2) Wang,Ci J.; Tseng,HW; Chou , C. L; Tsai, Τ·H.; Chen, C. Τ·; Lu, Μ. K. The vasorelaxation of Antrodia camphorata mycelia: involvement of endothelial 26 200825100

Ca(2+)-NO-cGMP pathway. Life Set 2003, 73,2769-2783. (3) Hseu, YC; Yang, HL; Lai, YC; Lin? J. G; Chen, G W.; Chang, YH Induction of apoptosis by Antrodia camphorata in human premyelocytic leukemia HL-60 cells. Nutr. O/Lu 2004, he 189-197· (4) Hsu, Υ·L·; Kuo, Υ·C·; Kuo, RL·; Ng, L· Τ·; Kuo, Υ·Η·; Lin, C· C. Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines. Cancer Lett. 2005,227, 77-89.

(5) Shen? YC; Chou, CJ; Wang5 YH; Chen, C. R; Chou, YC; Lu, Μ. K. Anti-inflammatory activity of the extracts from mycelia of Jnfrotfta cramp/zoraia cultured with water-soluble fractions From five different Cinnamomum species. FEMS Microbiol. Lett 20049 231,137-143. (6) Song, TY; Yen, G C. Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCU-induced hepatic toxicity in rats. J. Agric Food Chem, 2003, 57? 1571-1577 (7) Hoog, J. 0·; Hedberg, J. J.; Stromberg, P.; Svensson, S. Mammalian alcohol dehydrogenase- functional and structural implications. J. Biomed Sci 2Qdl, 8, 71-76· (8) Fliegmann, J·; Sandermann, H. Jr. Maize glutathione-dependent formaldehyde dehdrogenase cDNA: Plant Mol Biol 1997, 843-854. (9) Hess, DT; Matsumoto, A Kim, SO; Marshall, Η. E.; Stamler, JS Protein S-nitrosylation: purview and parameters. Nat Rev Mol Cell Biol 2005, 150-166. (10) Liu, L·; Hausladen, A·; Zeng ,M·; Que,L·; Hei Tman, J.; Stamler, JS A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature 2001, 410f 490-494. (11) Liu, L.; Yan, Y.; Zeng5 M.; Zhang, J. Hanes, MA; Abeam, G.; McMahon, TJ; Dickfeld, T.; Marshall, Η. E.; Que? L. G; Stamler, JS Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell 2004 , 116, 617-628. (12) Godoy, L.; Gonzalez-Duarte, R.; Albalat, R. S-nitrosogluthathione reductase activity of amphioxus ADH3: insights into the nitric oxide metabolism. Int. J. Biol Sci. 2006 , 2, 117-124. 27 200825100 (13) Uotila, L.; Koivusalo, M. Formaldehyde dehydrogenase. Methods Enzymol 1981, 77, 314-320. (14) Sanghani, P. C.; Robinson, H.; Bennett -Lovsey, R.; Hurley, TD·; Bosron, WF. * Structure-function relationships in human Class III alcohol dehydrogenase (formaldehyde dehydrogenase). Chemico-Biological Interactions 143-144^ 195-200. (15) Sakamotoa, A. Uedab, M.; Morikawaa, H. Arabidopsis glutathione-dependent for FEMS Letters 2002, 515, 20-24. [Simplified Schematic] Sequence Listing: 1 : A formaldehyde, alcohol and reduced nitration protein (GFD) The nucleic acid sequence has a full length of 1373 bp; the transcribed region is U34bp; and the coding region is 378 amino acid residues. · Description of the figure: • The birth number (*) is the transcription termination signal. 28

Claims (1)

200825100 X. Applying for a patent garden: 1. A kind of egg white (GFD) selected from the group consisting of anthraquinone and formaldehyde, alcohol and reduced nitration. The GHD of Ganoderma lucidum contains the amino acid sequence shown in Table 1. Two nucleic acids selected from the group consisting of Antrodia camphorata having both a de-alked pathway, alcohol and reduced nitrated egg protein (GFD), comprising a nucleic acid sequence which can be encoded as described in the scope of the patent application. Chito # GFD amino acid sequence. 3. According to the second paragraph of the patent application scope, the isolated nuclear nucleus of the Ganoderma lucidum GFD is expressed and quantified in the yeast. 4. A performance vector selected from the group consisting of an anthraquinone, an alcohol, and a reduced nitration eggshell (GFD), comprising a nucleic acid sequence of an isolated nucleic acid of Antrodia camphorata GFD as described in claim 2 of the patent application. #5. A host cell of Ganoderma lucidum GFD comprising the expression vector of Ganoderma lucidum GFD as described in claim 4 of the patent application. 6. A method for producing a protein of Antrodia camphora GFD, which is used for producing a protein of Antrodia camphora 6?0 according to Item 1 of the patent application scope, the method comprising the following steps: obtaining a Ganoderma lucidum GFD a nucleic acid sequence, which can be used to encode an amino acid sequence of the protein of the Ganoderma lucidum GFD; the nucleic acid sequence of the Ganoderma lucidum GFD is loaded into a expression vector; and the expression vector of the Ganoderma lucidum GFD is loaded into a host cell; The host cell of the Ganoderma lucidum GFD is cultured in a culture solution 29 200825100, which can allow the protein I encoded by the nucleic acid molecule of the Ganoderma lucidum GFD to synthesize the protein of the Ganoderma lucidum GFD; and the host cell and the culture solution The protein of the 樟 邙 邙 is recovered. 7. A pharmaceutical composition of Ganoderma lucidum GFD comprising the Ganoderma lucidum GFD protein as described in claim 1 of the patent application. 8. The pharmaceutical composition of Ganoderma lucidum GFD according to item 7 of the patent application scope wherein the Ganoderma lucidum GFD2 pharmaceutical composition is selected for use in health foods, cosmetics, scalding ointments and skin grafts. 9. A pharmaceutical composition of Ganoderma lucidum GFD according to item 8 of the scope of the patent application: wherein the health food prepared from the pharmaceutical composition of Ganoderma lucidum GFD is used for removing formaldehyde, alcohol and reducing nitration. 10. The pharmaceutical group according to claim 8 of the patent application scope, wherein the scald made of the pharmaceutical composition of the Ganoderma lucidum GFD is used to accelerate wound healing. U. The pharmaceutical composition according to the first aspect of the patent application, wherein the pharmaceutical composition of the GFD for skin transplantation is to prevent tissue necrosis of the transplanted skin by the GFD protein of the sputum, thereby accelerating the suturing of the transplanted skin . Things 30